This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes of the carotenoid biosynthesis pathway in plants and seeds.
Plant carotenoids are orange and red lipid-soluble pigments found embedded in the membranes of chloroplasts and chromoplasts. In leaves and immature fruits the color is masked by chlorophyll but in later stages of development these pigments contribute to the bright color of flowers and fruits. Carotenoids protect against photoxidation processes and harvest light for photosynthesis. The carotenoid biosynthesis pathway leads to the production of abscisic acid with intermediaries useful in the agricultural and food industries as well as products thought to be involved in cancer prevention. (Bartley, G. E., and Scolnik, P. A. (1995) Plant Cell 7:1027-1038).
Phytoene desaturase transforms phytoene to zeta carotene via phytofluene. cDNAs encoding this bifunctional enzyme have been identified from bacteria, peppers, corn, Arabidopsis and Narcissus. The lightly colored zeta-carotene is converted to neurosporene by the zeta-carotene desaturase enzyme (carotene 7, 8 desaturase; EC 1.134.99.30). cDNAs encoding zeta carotene desaturase have been identified in bacteria, corn, Narcissus, tomato, Arabidopsis and pepper. Neurosporene is further desaturated into lycopene. Lycopene may have one of two different fates: through the action of lycopene epsilon cyclase it may become alpha carotene, or it may be transformed into beta carotene by lycopene cyclase. Beta-carotene dehydroxylase converts beta-carotene into zeaxanthin. Zeaxanthin epoxidase transforms zeaxanthin into violxanthin and eventually absisic acid.
Zeaxanthin is the bright orange product highly prized as a pigmenting agent for animal feed which makes the meat fat, skin, and egg yolks a dark yellow (Scott, M. L. et al. (1968) Poultry Sci. 47:863-872). Gram per gram, zeaxanthin is one of the best pigmenting compounds because it is highly absorbable. Yellow corn, which produces one of the best ratios of lutein to zeaxanthin contains in average 20 to 25 mg of xanthophyll per kg while marigold petals yield 6,000 to 10,000 mg/kg.
The instant invention relates to isolated nucleic acid fragments encoding carotenoid biosynthetic enzymes. Specifically, this invention concerns an isolated nucleic acid fragment encoding a zeta carotene desaturase or a phytoene desaturase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding zeta carotene desaturase or phytoene desaturase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a carotenoid biosynthetic enzyme selected from the group consisting of zeta carotene desaturase and phytoene desaturase.
In another embodiment, the instant invention relates to a chimeric gene encoding a zeta carotene desaturase or a phytoene desaturase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a zeta carotene desaturase or a phytoene desaturase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a zeta carotene desaturase or a phytoene desaturase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a zeta carotene desaturase or a phytoene desaturase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a zeta carotene desaturase or a phytoene desaturase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of zeta carotene desaturase or phytoene desaturase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a zeta carotene desaturase or a phytoene desaturase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a zeta carotene desaturase or a phytoene desaturase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a zeta carotene desaturase or a phytoene desaturase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of zeta carotene desaturase or phytoene desaturase in the transformed host cell; (c) optionally purifyring the zeta carotene desaturase or the phytoene desaturase expressed by the transformed host cell; (d) treating the zeta carotene desaturase or the phytoene desaturase with a compound to be tested; and (e) comparing the activity of the zeta carotene desaturase or the phytoene desaturase that has been treated with a test compound to the activity of an untreated zeta carotene desaturase or phytoene desaturase, thereby selecting compounds with potential for inhibitory activity.